Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

successful use of bevacizumab in reversing anterior segment neovascularization and decreasing IOP 48 hours after a single intravitreal injection. Grisanti and colleagues (2006) noted that intracameral bevacizumab led to decreased vascular leakage in patients with NVG 1 day after injection. Further studies are needed to better understand the clinical role and safety profile of bevacizumab for intraocular injections.

Anti-neovascular medications may also have a role in preventing scarring after trabeculectomy or bleb needling procedures. Kahook and colleagues (2006c) reported the successful use of bevacizumab in a bleb needling procedure after repeated mitomycin C needling had failed. They noted a rapid decrease in neovascularization surrounding the bleb and an increase in bleb height and surface area. While promising, this was only a single case report that requires further corroboration to better understand the utility of bevacizumab in trabeculectomy surgery.

Other antiangiogenesis drugs are currently under investigation including ATG003 (Athenagen, San Francisco CA), a novel anti-angiogenic agent that inhibits endothelial nicotinic acetylcholine (nACh) receptors, selective integrin antagonists, pigment epithelium derived factor, and modified corticosteroids, and are all under investigation for treatment of ocular neovascular disease, but remain unproven. While most are being studied for age-related macular degeneration, some may find a role in treating anterior segment diseases.

VIII. THE FUTURE

BOX 16.1

This short review of current and future innovations is just a glimpse of what we can expect in the future. New developments will allow pharmaceutical

companies to produce designer drugs that target specific cells and avoid undesirable affects on healthy tissues. Clinical use of viral vectors to incorporate cellular changes on the genomic level may become a reality, with recent advances showing promise. Use of dendrimers, hyperbranched synthetic macromolecules that are modifiable in both size and structure, will allow for specific cell receptor targeting as dictated by surface functional groups. Dendrimers can possess both hydrophilic and hydrophobic properties making efficient lipophilic drug delivery, protected in an internal void space, a distinct possibility (Shaunak et al., 2004).

The future appears bright for new therapeutic discoveries and continued evolution towards advancing anterior segment ocular surgery and improving outcomes for patients.

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In the normal human eye the vitreous gel is bordered anteriorly by the posterior lens capsule, then the pars plana and pars plicata of the ciliary body, and posteriorly by the inner limiting membrane of the neural retina. The vitreous is an aqueous medium, composed mainly of water (98–99%) with smaller amounts of positively charged collagen stabilized by negatively charged hyaluronic acid. Over 50 other proteins have been identified in smaller amounts within the vitreous through the use of proteomics. Currently the surgical removal of vitreous gel, which is necessary for a broad spectrum of disease processes, is performed by mechanical means, and little use has

surgery. However, enzyme assisted posterior segment surgery has many theoretical and practical advantages over mechanical vitrectomy, including reducing operating time and reducing complications that typically occur during mechanical vitreous separation of the posterior hyaloid from the inner retinal surface. Herein we describe the enzymatic agents that have been proposed for vitreous surgery, including their current status, and advantages and disadvantages of each agent, with an emphasis on the barriers to clinical use. The goal is to give the reader a complete understanding of therapeutic agents that are likely to be available in the near future for posterior segment vitreous surgery.

With the advent of modern vitreoretinal surgery in the 1970s surgeons were given a set of mechanical instrumentation for physically removing the vitreous and its associated intraocular pathology (Machemer, 1976a,b,c; Machemer and Allen, 1976). For the most part, vitreoretinal surgery over the next three decades involved use of physical instruments to repair and restore altered intraocular anatomy. This involved the removal of the vitreous via mechanical means, with additional steps that were dependent upon the pathology that was being corrected. For example, surgery for macular pucker involved mechanical removal of the vitreous gel, mechanical separation of the epiretinal membrane from the retinal surface, and then mechanical removal of the membrane from the eye (Machemer, 1976a,b,c; Machemer and Allen, 1976). Similarly, removal of vitreous hemorrhage or severe intraocular inflammation involved mechanical removal of the vitreous debris (blood and inflammatory cells) to clear the intraocular fluid and allow for return of visual function (Machemer, 1976a). Repair of retinal detachment involved either external scleral buckling with little manipulation of the vitreous gel or vitrectomy surgery, in which mechanical removal of the vitreous was combined with either laser photocoagulation or cryotherapy to induce chorioretinal adhesion, and installation of gas or silicone oil for intraocular tamponade (Michels, 1976, 1977, 1978, 1979, 1981, 1984a, 1984b; Michels et al., 1974, 1983).

A common thread in these procedures is removal of the vitreous gel through mechanical means. Although there have been occasional case series describing repair of macular pucker, macular holes and other conditions, without removal of the bulk of the vitreous gel, for the most part a transvitreal approach to ocular disease requires vitreous gel removal to minimize retinal tears, post-operative transvitreal traction, and other complications.

At the current time surgical removal of the vitreous gel is accomplished by mechanical

means, in which mechanical forces are imparted via aspiration and cutting with a vitrector, and perpendicular and tangential sheer forces are transmitted via the use of vitreoretinal picks and forceps to manipulate intraocular tissue. Future advances in vitreoretinal surgery are likely to involve significant changes in the treatment paradigm, with the use of pharmacological agents for vitreous gel removal. Herein we will describe the normal anatomy of the vitreous gel and its composition, with an emphasis on the anatomy that is most important to posterior segment ocular surgery, such as vitreous adhesion to adjacent anatomic structures. We will discuss normal vitreous anatomy and composition, with attention to the aspects of molecular composition that play an important role in the physical and chemical properties of the vitreous gel. We will then discuss therapeutic agents to facilitate posterior segment ocular surgery, with an emphasis on the vitreolytic agents that are likely to assist vitreoretinal surgeons in the management of the posterior vitreous diseases in the future.

II. NORMAL VITREOUS ANATOMY

AND COMPOSITION

The vitreous gel in the normal human eye is bordered by the posterior lens capsule anteriorly, by the pars plana and pars plicata of the ciliary body, and posteriorly by the internal limiting membrane of the retina. At the optic nerve head there is a condensation of vitreous fibrils that is most evident upon the development of anatomic separation of the vitreous from the retina (Weiss’ ring). The adhesion between the vitreous and posterior lens capsule is particularly strong, making it difficult to separate these two structures without causing lens damage in the normal phakic eye. A second area of strong adhesion is along the vitreous bases, which is a zone of condensed vitreous fibrils that straddles the ora serrata; it extends approximately 2mm anterior and

2–4mm posterior to the ora serrata, depending upon the meridian. During posterior vitreous detachment, which is a normal aging process in the eye, the vitreous separates from the internal limiting membrane of the retina by vitreous liquefaction (synchisis) with secondary collapse of the vitreous body. Central vitreous liquefaction induces separation of the posterior hyaloid surface of the vitreous from the internal limiting membrane (syneresis). During this latter process patients can develop retinal tears, vitreous hemorrhage, and/or retinal detachment due to the tight adhesion of the posterior hyaloid along the major vessels; there is also typically pathological adherence in regions of lattice deceleration, which can predispose patients to retinal tears and detachment (Burton, 1989; Folk et al., 1989, 1990; Markham and Chignell, 1977; Tillery and Lucier, 1976). Over the last two decades there has been an evolving understanding of the role of the vitreous in maintaining ocular health, including the role of vitreous in maintaining lens clarity by acting as a “sink” to lower oxygen tension at the posterior lens surface (Barbazetto et al., 2004).

In the normal human eye the vitreous body is composed predominantly of water (98–99%) with smaller amounts of collagen and hyaluronic acid. These two molecules give the vitreous structural support, as positive charges along the collagen fibers are stabilized by the negatively charged hyaluronic acid molecules. Other glycosaminoglycans exist in vitreous, which interact with the collagen fibrils via noncovalent bonds. The adhesion between the posterior vitreous surface and the internal limiting membrane depends upon the presence of laminin, fibronectin, glycoconjugates, and collagen Types I and IV (Russell et al., 1991). The concentration of hyaluronic acid decreases as a function of age, typically starting at around age 50 (Hayreh and Jonas, 2004); although the reasons for this are incompletely understood, the loss of hyaluronic acid may be responsible for destabilization of the vitreous which

causes synchisis followed by syneresis. To a large extent this is a normal process, occurring in most individuals between the ages of 50 and 70 (Hayreh and Jonas, 2004).

During embryonic development the hyaloidal vasculature temporarily nourishes the posterior surface of the lens and primary vitreous and then regresses; proteomic analysis suggests that this occurs due to activin receptor-like kinase-1 (ALK1), a type I receptor for transforming growth factor-beta1 (Albe et al., 2005) The complete proteome of the vitreous is not yet identified; dynamic light scattering suggests that hyaluronic acid concentrations increase from anterior to posterior, with the reverse true for viscosity, implying that the lower concentration of hyaluronan near the lens is offset by increased molecular size. Concentration gradients are also seen in 6 nm diameter and 15 nm diameter particles whose identity is yet to be determined via proteomics (Bettelheim and Zigler, 2004). Pathological conditions lead to increases in the concentrations of various proteins in the vitreous. For example, in diabetic macular edema there is an increase in PEDF, ApoA-4, ApoA-1, Trip-11, PRBP, and VDBP (Ouchi et al., 2005). Fifty-two proteins have been seen in gels from human vitreous and 35 of these proteins were not seen in plasma (Ouchi et al., 2005). Pigment epithe- lium-derived factor, which was reported to be a potent inhibitor of angiogenesis in cornea and vitreous, was present at a higher concentration in vitreous hemorrhage due to diabetes than in patients with macular hole (Ouchi et al., 2005). In the future proteomics will shed further light on the composition of vitreous in the normal and diseased human eye (Shimizu et al., 2002).

III. IMPORTANCE OF POSTERIOR

VITREOUS DETACHMENT

As mentioned above, the posterior hyaloid surface of the vitreous gel is adherent to the inner retinal surface in the normal

human eye, most prominently at the vitreous base, optic disc and along the major retinal vessels (Nishikawa and Tamai, 1996). The development of a posterior vitreous detachment will sometimes leave small islands of cortical vitreous attached to the inner retinal surface (Nishikawa and Tamai, 1996). During vitreous surgery it is often necessary to detach the posterior hyaloid from the internal limiting membrane of the retina; currently this is done mechanically with aspiration or the use of a spatula. However, mechanical separation of the posterior hyaloid from the internal limiting membrane is associated with iatrogenic retinal breaks or hemorrhage, and incomplete removal of posterior hyaloid may provide a surface for postoperative cellular proliferation. Separation of the posterior hyaloid from the retinal inner limiting membrane has an impact on the natural course of many vitreoretinal diseases, including diabetic retinopathy (Hendrikse and Yeo, 1993; Tagawa et al., 1986; Yonemoto et al., 1994), vitreomacular traction syndrome (Smiddy et al., 1988), idiopathic macular holes (Hikichi et al., 1993) and retinal detachment. It is often necessary to remove the posterior hyaloid during pars plana vitrectomy, especially in cases of macular hole, penetrating ocular trauma and proliferative diabetic retinopathy (Bonnet, 1988; Gregor and Ryan, 1983). The use of small gauge vitrectomy (25 and 23 gauge) makes this separation more difficult to achieve with vitrector aspiration alone (Eckardt, 2005; Ibarra et al., 2005).

All vitrectomy surgery, regardless of the instrumentation gauge, would be facilitated by the availability of pharmacological agents to separate the posterior hyaloid from the neural retina in a safe and atraumatic fashion. Pharmacologically induced posterior vitreous detachment could drastically improve safety profiles for vitrectomy and increase the indications for vitreous surgery. In addition, pharmacological induction of a posterior vitreous detachment may have considerable use

in patients with diabetes who are at risk of developing proliferative diabetic retinopathy, as benefit can be obtained from inducing molecular vitreous separation before developing this advanced stage of the disease. Vitreous detachment may also improve visual acuity in select patients with other retinal disorders of the vitreomacular interface, such as vitreomacular traction and macular hole. For these reasons, several workers have attempted to induce a posterior vitreous detachment prior to or during vitreous surgery by using intravitreal injection of different enzymes (Moorhead et al., 1980; O’Neill and Shea, 1973; Pirie, 1949; Verstraeten et al., 1993), homologous blood (Squire and McEwen, 1958), an expansile gas (Chan et al., 1995), or intraocular diathermy (Vander and Kleiner, 1992). However, none of these earlier techniques led to specific cleavage of the binding sites between the internal limiting membrane and the posterior hyaloid surface.

Several drugs are available for intraocular induction of a posterior vitreous detachment (Sebag, 2005). In general, these drugs act by one of two mechanisms: vitreous liquefaction, in which the drug causes central liquefaction of the vitreous gel, collapse of the vitreous body, and secondary separation of the posterior hyaloid from the neural retina; and targeted enzymatic posterior vitreous detachment, in which the enzyme selectively cleaves the anatomic attachment between the posterior hyaloid and the inner surface of the retina (Figure 17.1). Most drugs used for pharmacological vitreolysis fall into the first category; to our knowledge, the only drug that specifically cleaves the attachment between the posterior hyaloid and the internal limiting membrane is dispase, which is a neutral bacterial protease that has a selective action against fibronectin and collagen IV. In principle vitreous liquefaction is more hazardous than specific enzymatic cleavage of the posterior vitreous from the inner retina, because vitreous liquefaction with

secondary vitreous detachment collapse would be expected to carry a higher rate of iatrogenic retinal tears. Herein we will review the current status of several agents that have been used to induce vitreous separation, and discuss the obstacles that must be overcome for widespread use of these agents to assist with therapeutic removal of the posterior vitreous gel.

IV. SPECIFIC AGENTS FOR

POSTERIOR SEGMENT VITREOUS

REMOVAL

A. Plasmin

Plasmin is an autologous serum protease that is a key component of the fibrinolysis cascade. Plasmin is a non-specific protease usually present in human serum, and it is responsible for degrading a variety of plasma proteins; its specific physiologic role is to degrade fibrin clots. Plasmin is created when plasminogen, its precursor, is released into the circulation and activated by tissue

plasminogen activator (TPA), urokinase plasminogen activator (uPA), or streptokinase. The activity of plasmin can be inhibited by the presence of alpha 2-antiplasmin, a serine protease inhibitor. Deficiency in plasmin may lead to thrombosis, as clots are not degraded adequately. Plasmin is not present in normal vitreous, but is present in the subretinal fluid of patients with rhegmatogenous retinal detachment; prior authors have speculated that the presence of plasmin may increase detachment of the retinal pigment epithelium from the inner aspects of Bruch’s membrane and thus accelerate or increase the risk of proliferative vitreoretinopathy (Immonen et al., 1989, 1988).

Several approaches have been used to induce posterior vitreous detachment with plasmin. Gandorfer et al. (2004) injected plasmin (1-2 U/100 microliters) into the vitreous of enucleated porcine eyes, and showed that eyes receiving plasmin had separation of the cortical vitreous from the internal limiting membrane with no structural changes in the retina, with the degree of separation depending on the concentration

and duration of plasmin exposure (Gandorfer and Kampik, 2005). Plasmin has been injected into the vitreous cavity of rabbits in vivo and been shown to be vitreolytic without toxicity (Kim et al., 2004). Intravitreal plasmin also induces a posterior vitreous detachment in human eyes in vitro after intravitreal injection (Li et al., 2002). Prior workers have used TPA as a biological activator to convert plasminogen to plasmin in vivo; predominantly TPA has been used to lyse blood clots in this setting, but TPA has also been used to induce a posterior vitreous detachment with simultaneous addition of cryotherapy to break down the blood–retinal barrier and therefore allow plasminogen to move into the vitreous cavity (Hesse et al., 1995, 2000). Hikichi et al. (1999) induced a posterior vitreous detachment with a combination of intravitreal injection of sulfur hexafluoride plus intravitreal plasmin injection to induce a posterior vitreous detachment; it is possible that either alone would have induced a posterior vitreous detachment in this experimental setting.

In the clinical setting we can envision several ways in which plasmin can be used to induce vitreous liquefaction followed by vitreous detachment. Autologous plasmin can be harvested from a patient’s blood prior to surgery, purified, and then injected into the vitreous cavity (Asami et al., 2004; Azzolini et al., 2004). An affinity cartridge has been developed so that autologous plasminogen can be used for posterior vitreous detachment induction (Asami et al., 2004; Azzolini et al., 2004). The plasminogen is then converted to plasmin using streptokinase, which can then be used for surgical procedures. The method can be adapted to purify other blood components. Autologous plasmin enzyme has been demonstrated to assist with the production of posterior vitreous detachment in patients undergoing surgery for diabetic macular edema; the level of suction required during vitreous surgery to induce a posterior vitreous detachment was lower in the plas- min-treated versus control eyes (Asami

et al., 2004; Azzolini et al., 2004). Plasmin has also been used as a surgical adjuvant for the closure of traumatic (Chow et al., 1999) and pediatric macular holes (Margherio et al., 1998). Autologous plasmin enzyme has been used during diabetic vitrectomy for macular edema, and been shown to create a posterior vitreous detachment in this setting and thereby facilitate surgery (Sakuma et al., 2006, 2005a). A clinical trial of this agent is currently ongoing. Intravitreal injection of TPA coupled with cryotherapy has been demonstrated to be efficacious for this purpose (Hesse et al., 1995, 2000).

B. Microplasmin

Microplasmin is a truncated form of plasmin; microplasmin contains the active site of plasmin and has a similar mechanism of action in vitreolysis. Microplasmin was initially produced by cleavage of plasmin (Wu et al., 1987a,b), but recombinant microplasmin has been produced (Medynski et al., 2006; Nagai et al., 2003). Microplasmin is being tested in a Phase IIb clinical trial to determine the safety and efficacy of intravitreal microplasmin in facilitating the creation of a posterior vitreous detachment. The drug (or placebo) will be injected 7 days prior to planned pars plana vitrectomy in patients with no posterior vitreous detachment. The endpoint is the presence of a spontaneous posterior vitreous detachment at 7 days, or a decrease in the amount of vitrector suction required to induce a posterior vitreous detachment during surgery. Waiting 7 days may be required due to the mechanisms of action of the drug, which is known to induce syneresis after inducing synchysis (Figure 17.1).

Microplasmin has received orphan drug status by the Food and Drug Administration for its use during pediatric vitreoretinal surgery. Use of microplasmin avoids the issues associated with preparation of autologous plasmin and is expected to lead to a reduction in the amount of suction

required to achieve posterior vitreous detachment. Unlike TPA, microplasmin is a direct acting thrombolytic, as compared to most other thrombolytics which dissolve clots indirectly by activating the plasmin precursor, plasminogen. Microplasmin may have neuroprotective features and have a reduced risk of bleeding compared to indi- rect-acting thrombolytics (Lapchak et al., 2002; Suzuki et al., 2004). Once microplasmin enters into the systemic circulation, it is rapidly inactivated by a blood protein (alpha-2 anti-plasmin) thus reducing the risk of bleeding in locations away from the intended treatment area.

Microplasmin has been shown to induce a posterior vitreous detachment in donor human eyes in vitro and feline eyes in vivo. (Gandorfer et al., 2004). Thirteen human eyes were incubated with escalating doses of microplasmin, with four of the eyes receiving simultaneous intravitreal gas injection (Gandorfer et al., 2004). In all control eyes, scanning electron microscopy demonstrated there was cortical vitreous covering the inner limiting membrane, but intravitreal injection of 125 or 188 micrograms of microplasmin resulted in complete posterior vitreous detachment; lower doses were not sufficient to separate the cortical vitreous even if a gas injection was also given. In cat eyes in vivo 25 micrograms of microplasmin resulted in complete posterior vitreous detachment after 3 days, with complete posterior vitreous detachment within 3 weeks at lower doses. The retina and the internal limiting membrane were well preserved in all eyes (Gandorfer et al., 2004; Sakuma et al., 2005b; Sebag, 2005; Sebag et al., 2006). In the feline eye, there is no cellular response of retinal glial cells or neurons. Intravitreal injection of recombinant microplasmin in the rabbit induces no ERG or retinal ultrastructural abnormalities (Sakuma et al., 2005b). Thus microplasmin induces a dose-dependent cleavage between the vitreous cortex and the internal limiting membrane without morphologic alterations in the retina.

The effect of microplasmin on vitreous diffusion coefficients was investigated using dynamic light scattering. Dynamic light scattering performed after injections of human recombinant microplasmin into the vitreous of porcine eyes reveals that microplasmin increased porcine vitreous diffusion coefficients of injected 20 nm microspheres in a dose-dependent manner (Sebag, 2005; Sebag et al., 2006).

C. Hyaluronidase

As mentioned above, the aim of using enzymes in vitreoretinal surgery is to induce or facilitate posterior vitreous detachment and thus assist with pharmacological vitrectomy. This can be achieved by liquefying the gel structure of the vitreous (synchisis) with secondary collapse of the vitreous gel and induction of posterior vitreous detachment, or by directly weakening the adherence of the posterior vitreous cortex to the retina (syneresis) (Czajka and Pecold, 2002). For substrate-specific enzymes, the ability of various enzymes to induce a posterior vitreous detachment depends on the composition of the vitreous and its attachment to the internal limiting membrane, but the molecular composition of the vitreous is less important for the action of non-specific proteases like plasmin and microplasmin (Figure 17.1).

In contrast to plasmin or microplasmin, hyaluronidase is a substrate-specific enzyme that induces synchisis by acting on proteoglycans in the vitreous. Intravitreal injection of 1 IU of intravitreal hyaluronidase is sufficient for partial vitreolysis and is non-toxic to the rabbit retina (Gottlieb et al., 1990). Intravitreal injection of hyaluronidase in doses of 10 IU or higher induces posterior vitreous detachment in rabbits over a period of 5 weeks. Intravitreal doses of 20 IU or less do not appear to affect the microscopic morphology or function of ocular structures adversely. Injections of hyaluronidase, therefore, could be considered as an alternative or adjunct